Crosslinking agents and methods of use

ABSTRACT

Polymeric crosslinking agents are disclosed that have an inert water soluble polymeric component, biodegradable components, functional components reactive with chemical groups on a protein, for example, amine or thiol groups. The inert polymeric component may be flanked at each end with a biodegradable component which is flanked at each end with a protein reactive functional component. A polymeric crosslinking agent is disclosed having a biodegradable component, polyalkylene oxide, and at least three reactive functional groups that are each capable of forming a covalent bond in water with at least one functional group such as an amine, thiol, or carboxylic acid.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No. 1009/147,897 filed Aug. 30, 1999 entitled “Methods and Devices forPreparing Protein Concentrates” filed under 35 U.S.C. §3.71 withpriority to International Application No. PCT/US97/16897, filed Sep. 22,1997, which claims priority to U.S. application Ser. No. 60/026,526filed Sep. 23, 1996; U.S. application Ser. No. 60/039,904 filed Mar. 4,1997 and U.S. application Ser. No. 60/040,417 filed Mar. 13, 1997, thedisclosures of which are herein incorporated by reference.

TECHNICAL FIELD

The field of the invention is synthetic and natural molecules used tomake polymers for treating patients.

BACKGROUND OF THE INVENTION

Methods of preparing concentrated protein compositions from initialdilute protein compositions find use in a variety of differentindustries, including the chemical, biological, academic research,biotechnological and medical industries. For example, “Fibrin Sealants”(also known as fibrin gels or fibrin glues) are a type of blood derivedcomposition used in the medical industry which are prepared throughmethods of concentrating blood plasma proteins that have been developedfor use as tissue adhesives, drug delivery vehicles and the like.Although such compositions are not yet FDA approved in the United Statesdue to concerns over blood borne contaminants, such compositions aremarketed in Europe and elsewhere throughout the world. A typicalcommercial fibrin glue kit consists of a vial of lyophilizedconcentrated human fibrinogen, prepared from pooled human donor blood,that also contains fibronectin, Factor XIII and reduced amounts ofplasminogen. The concentrate, also known as cryoprecipitate, isreconstituted with a reconstituting solution and warmed to 37° C. Thesecond component of the adhesive system is a lyophilized bovine thrombinsolution which is reconstituted with a calcium chloride solution. Theformulation may also contain additional components like a fibrionolysisinhibitor. The reconstituted solutions are mixed and used as a surgicaladhesive system.

The most common method used for the preparation of the fibrinogencomponent of the above described kits is cryoprecipation. Incryoprecipitation, fresh blood plasma is frozen at −80° C. for at least6 to 12 h. The temperature of the frozen plasma is then raised to around0-4° C., resulting in the formation of a precipitated supernatant thatcontains fibrinogen and Factor XIII, i.e. a cryoprecipitate. Thecryoprecipitated suspension is then recovered. Another method describedin the literature is the use of common non-toxic organic/inorganiccompounds such as ethanol, polyethylene glycol, poly(vinyl alcohol),1-6-hexanoic acid and ammonium sulfate as precipitating agents.

The above methods of preparing the fibrinogen containing component offibrin glue compositions are time consuming and complex. Furthermore, inapproaches such as cryoprecipitation, special equipment like arefrigerated centrifuge, is often required. Finally, different methodsof precipitation produce fractions with different adhesive and physicalcharacteristics which can adversely affect the ultimate adhesiveproduct.

Accordingly, there is a continued need for the development of newmethods for preparing concentrated protein compositions, andparticularly fibrinogen rich fractions from blood compositions. Ideally,such methods would: be relatively simple and rapid; require minimalhandling of the plasma and not include a cryoprecipitation step; andprovide serum concentrates suitable for use in fibrin glue systems, inwound healing promotion systems, in drug delivery, and in tissueregeneration. Furthermore, such methods would ideally be suitable foruse in the preparation of autologous serum concentrates that eliminatepathogen transmission risk present in serum concentrates prepared frompooled donor sources. Also of interest would be the development of asimple method capable of efficiently producing concentrated proteincompositions from large volumes of initial fluid, e.g. pooled humanserum in emergency surgery situations. Also of interest would be thedevelopment of devices for use in performing the subject methods.

Relevant Literature

Fibrin sealants and methods for their production, as well as clinicalapplications thereof, are reviewed in David H. Sierra, “Fibrin SealantAdhesive Systems: A Review of Their Chemistry, Material Properties andClinical Applications,” J. Biomaterials Applications (1993) 7:309-352.Other references of interest include: Sierra & Feldman, J. AppliedBiomaterials (1992) 3:147-151; U.S. Pat. Nos. 5,405,607; 5,030,215; and5,395,923.

Devices for preparing and administering a fibrin sealant to facilitatetissue repair are described in: U.S. Pat. Nos. 4,874,368; U.S. Pat. No.4,631,055; U.S. Pat. No. 4,735,616; U.S. Pat. No. 4,359,049; U.S. Pat.No. 4,978,336; U.S. Pat. No. 5,116,315; U.S. Pat. No. 4,902,281; U.S.Pat. No. 4,932,942; WO 91/09641, and Tange, R. A., Fibrin Sealant inOperative Medicine: Otolaryngology-Vol. 1 (1986).

Microencapsulated drug particles and similarly protectedpharmaceutically active agents are described in: Kissel et al., J.Controlled Release (1991) 16:27; Tabata et al., Pharmaceutical Research(1993) 10:487; EPA 83303606.4; U.S. Pat. No. 5,143,662; Mathiowitz &Langer, J. Controlled Release (1987) 5:13; Nihant et al., J. Colloid &Interface Science (1995) 173:55; and Irwin et al., PharmaceuticalResearch (1994) 11: 1968.

Hydrogels and methods for their preparation are reported in: U.S. Pat.Nos.: 5,626,863; 5,573,934; 5,567,435; 5,410,016; 5,529,914; 5,514,380;5,476,909; 5,041,292, 5,583,114; as well as in Walter et al., J.Macromol. Sci.-Phys. (1994) B33 (3&4):267; Pathak et al., J. Am. Chem.Soc. (1992) 114: 8311; Sawhney et al., Macromolecules (1993) 26:581;Keogh & Eaton, J. Laboratory & Clinical Med. (1994) 124:4:537; and Reddiet al., Macromolecular Reports (1995) A32:789.

SUMMARY OF THE INVENTION

Methods and devices for the preparation of protein concentrates, as wellas novel protein concentrate compositions prepared thereby, productsderived therefrom and applications in which the products find use, areprovided. In the subject methods, an initial protein comprisingcomposition, such as whole blood-or plasma, is contacted with anon-protein denaturant hydrogel under conditions sufficient for asubstantial portion of the water present in the initial composition tobe absorbed by the hydrogel. Following absorption, the resultant proteinrich phase is separated from the swollen hydrogel phase to produce theprotein concentrate. The subject methods find use in a variety ofapplications, particularly in the preparation of fibrinogen richcompositions from whole blood or derivatives thereof, where theresultant fibrinogen compositions find use in a variety of applications,as fibrin sealant tissue adhesives, as drug delivery vehicles, assources of growth factors to promote wound healing, and the like.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 provides a schematic representation of the inventive method ofpreparing fibrinogen rich compositions.

FIG. 2 provides a schematic representation of the preparation of a drugloaded fibrin gels according to the subject invention. In this figure,vial A contains controlled release microspheres encapsulated withbioactive compounds and calcium salt. Vial B contains all the componentsof a fibrin glue system (i.e. fibrinogen, thrombin etc.) except calcium,in a lyophilized form. Vial A and B are reconstituted with sterilesaline solution and used as a fibrin glue system. A and B are mixed andapplied in situ in a surgical field using a suitable surgical device.

FIG. 3 provides a graphical representation of the water absorptiveproperties of a PEG 20,000 diacrylate hydrogel.

FIG. 4 provides a graphical representation of the protein concentrationover time in a protein comprising aqueous composition contacted with ahydrogel according to the subject invention.

FIG. 5 provides a schematic representation of various polymericcrosslinking agents of the subject invention. In this figure, ()represents a biodegradable component such as polyhydroxy acids,polylactones and their copolymers, or synthetic peptide sequences whichare cleaved by enzymes inside the human body; () represents a reactivefunctional group such as carbodiimidazole, aldehyde, epoxide,n-hydroxysuccinimide and the like; () represents a biocompatible inertcomponent, such as polyethylene glycol, dextran, polyvinyl alcohol; ()represents a copolymer of trimethylene carbonate and lactones or asynthetic peptide sequence which is cleavable by human enzymes.

FIG. 6 depicts a first embodiment of a device for producing proteinconcentrates from a physiological fluid according to the subjectinvention.

FIGS. 7(a) to (e) depicts a second embodiment of a device for producingprotein concentrates from a physiological fluid according to the subjectinvention.

DESCRIPTION OF THE SPECIFIC EMBODIMENTS

Methods and devices for the preparation of protein concentrates, as wellas novel protein concentrate compositions prepared thereby and productsderived therefrom, are provided. In the subject methods, an initialprotein comprising composition, such as whole blood or plasma, iscontacted with a non-protein denaturant hydrogel under conditionssufficient for a substantial portion of the water present in the initialcomposition to be absorbed by the hydrogel. Following absorption, theresultant protein rich phase is removed from the swollen hydrogel toproduce the protein concentrate, where the term “removed” is employed ina broad sense to mean that the swollen hydrogel and the protein richphase are isolated from one another, where removal can be accomplishedvia active separation, e.g. by centrifugation, or passively, e.g. inthose embodiments where the hydrogel and protein rich phase separate bythemselves, where the protein rich phase is subsequently isolated fromthe hydrogel through simple aspiration or other analogous technique. Byselecting an appropriate hydrogel, the nature of the resultant proteinconcentrate may be controlled. The subject methods find use in a varietyof applications, particularly in the preparation of fibrinogen richcompositions from whole blood or derivatives thereof (where the initialblood composition may be a pooled donor source or an autologous source,where the resultant fibrinogen compositions find use in a variety ofapplications, fibrin sealant tissue adhesives, drug delivery vehicles,and the like. In further describing the subject invention, the methodsfor preparing the subject protein concentrates will first be describedin greater detail, followed by a discussion of the resultantconcentrates themselves, representative applications in which they finduse and kits and devices suitable for use in their preparation.

Before the subject invention is further described, it is to beunderstood that the invention is not limited to the particularembodiments of the invention described below, as variations of theparticular embodiments may be made and still fall within the scope ofthe appended claims. It is also to be understood that the terminologyemployed is for the purpose of describing particular embodiments, and isnot intended to be limiting. Instead, the scope of the present inventionwill be established by the appended claims.

It must be noted that as used in this specification and the appendedclaims, the singular forms “a,” “an,” and “the” include plural referenceunless the context clearly dictates otherwise. Unless defined otherwiseall technical and scientific terms used herein have the same meaning ascommonly understood to one of ordinary skill in the art to which thisinvention belongs.

In practicing the subject methods, an initial protein comprisingcomposition is contacted with a hydrogel under conditions sufficient forat least a portion of at least the water present in said bloodcomposition to be absorbed by the hydrogel. The initial proteincontaining composition may be any aqueous composition that comprises oneor more proteins of interest, where such compositions include bothnaturally occurring compositions, such as physiologically derivedfluids, e.g. blood, plasma, urine, cerebrospinal fluid, tears, saliva,milk, peritoneal cavity fluid and the like; and synthetically preparedcompositions, e.g. tissue culture medium, and the like. Physiologicalfluids of interest may be obtained from a variety of hosts, includingcows, sheep, pigs, deer, humans and the like. For example, the subjectmethods can be used to produce enriched protein compositions from cow orsheep milk, where the cow or sheep may be a transgenic animal engineeredto produce milk containing a recombinant protein of interest.

Of particular interest is the preparation of protein concentrates frominitial blood compositions. The blood composition employed in thesubject methods will typically be derived from a mammalian source, wheresuitable sources include cows, sheep, pigs, deer, humans and the like,where humans may be the preferred source depending on the intended useof the composition. The blood composition may be whole blood or a bloodproduct, i.e. a whole blood derivative, where whole blood is subjectedto one or more filtration steps, e.g. removal of red blood cells, andthe like. In other words, blood compositions that may be used in thesubject invention include both whole blood and blood derivatives, e.g.plasma, platelet containing serum, and the like, where such bloodcompositions may be obtained from their hosts and produced according tomethods known in the art. The blood composition, prior to use, may bescreened for the presence of one more pathogens, e.g. AIDS, Hepatitis B,etc. One or more components may be added to the blood composition, suchas anticoagulants, and the like. The blood composition may be derivedfrom a pooled supply of blood from a variety of different hosts, i.e. apooled donor supply, or from the host in which the ultimate proteinconcentrate is to be employed, i.e. an autologous source.

For clarity and ease of understanding, the invention is furtherdescribed below in terms of the preparation of a protein concentrate,e.g. a fibrinogen rich composition, from an initial blood composition asdescribed above. However, in so describing the invention, it should beunderstood that the full scope of the invention also encompasses thepreparation of protein concentrates from non-blood compositions, asdescribed above.

The hydrogel with which the blood composition is combined in the subjectmethods may comprise one or more different hydrogels in combination,where usually no more than four different hydrogels, and more usually nomore than three hydrogels will be used together, where a combination ofdifferent hydrogels may be employed for greater control over the typesof components of the blood composition that are absorbed by the hydrogeland the nature of the resultant fibrinogen rich-composition. Forexample, a hydrogel blend may be employed that combines a hydrogel witha first particular cut-off value, as described in greater detail below,and a second hydrogel that selectively removes a particular component ofthe initial fluid, e.g. albumin. Hydrogels employed in the subjectmethods are compositions that are capable of absorbing water from anaqueous composition with which they are contacted to swell in size andincrease in mass, where the increase in mass will typically be at least10 and up to 1000 fold or greater than the dry mass of the hydrogel.Importantly, the hydrogels will be hydrogels which do not denature ormodulate the conformation of proteins with which they come into contact,i.e. the hydrogels employed in the subject invention are non-proteindenaturant hydrogels. Preferred are hydrogels that are highly resistantto protein adsorption.

Hydrogels suitable for use in the subject methods will be biocompatible,by which is meant that they are suitable for contact with a bloodderived composition that is to be introduced into a mammalian host, i.e.they will not leach toxic or unwanted substances into the bloodcomposition upon contact. Preferably, the hydrogel will be one that doesnot substantially change the pH of the blood composition with which itis contacted and is sterile in nature.

Suitable hydrogels include macromolecular and polymeric materials intowhich water and small molecules can easily diffuse and include hydrogelsprepared through the cross linking, where crosslinking may be eitherthrough covalent, ionic or hydrophobic bonds introduced through use ofeither chemical cross-linking agents or electromagnetic radiation, suchas ultraviolet light, of both natural and synthetic hydrophilicpolymers, including homo and co-polymers. Hydrogels of interest includethose prepared through the cross-linking of: polyethers, e.g.polyakyleneoxides such as poly(ethylene glycol), poly(ethylene oxide),poly(ethylene oxide)-co-(poly(propyleneoxide) block copolymers;poly(vinyl alcohol); poly(vinyl pyrrolidone); polysaccharides, e.g.hyaluronic acid, dextran, chondroitin sulfate, heparin, heparin sulfateor alginate; proteins, e.g. gelatin, collagen, albumin, ovalbumin orpolyamino acids; and the like. Because of their high degree ofbiocompatibility and resistance to protein adsorption, polyether derivedhydrogels are preferred, with poly(ethylene glycol) derived hydrogelsbeing particularly preferred.

Physical characteristics such as size, shape and surface area can affectthe absorption characteristics of the hydrogel composition. Accordingly,the hydrogel composition that is employed may be in a variety ofconfigurations, including particles, beads, rods, sheets, irregularshapes and the like, where those shapes with greater surface area tototal mass ratios are preferred, at least in certain embodiments. Theporosity of the hydrogel, which is dependent on the amount or degree ofcrosslinking present in the hydrogel, also affects the absorptioncharacteristics of the hydrogel. For example, where absorption of waterand small molecules is desired, a hydrogel with a high degree ofcrosslinking and a consequent small average porosity will be employed.Conversely, where the selective absorption of small proteins is alsodesired, a hydrogel with a low degree of crosslinking and a consequentlarge average porosity will be employed. Of interest for certainembodiments of the subject invention, e.g. where the selective removalof albumin and other components of similar or lower molecular weight isdesired, are hydrogels that have a molecular weight “cut-off” absorptionpoint (i.e. a molecular weight limitation in excess of which componentsare not absorbed) of about 80,000 daltons. Other hydrogels of particularinterest are for use in certain embodiments are those having molecularweight cutoffs of: 100,000 daltons (where one desires to retainimmunoglobulins); 150,000 daltons (where the removal of immunoglobulinsis desired); 15,000 daltons (where one desires to retain growth factorsin the protein concentrate); etc.

To further tailor the absorptive properties of the hydrogel, thehydrogel can be modified to provide for specific binding of one or moreof the components of the blood composition to the surface of thehydrogel. For example, where the selective absorption of albumin inaddition to water is desired, the hydrogel can be modified to comprisean albumin specific binding reagent, such as Cibacron blue, as describedin Keogh & Eaton, J. Lab. & Clin. Med. (1994) 124:537. Other means oftailoring the hyrdogels to further control the nature of the proteinconcentrate produced by the subject method include using hydrogelscomprising agents that act as water absorbents and/or precipitants,where such agents include ethanol, PEG 400, phosphate buffer and thelike.

The hydrogel compositions employed in the subject methods can beprepared by methods known to those skilled in the art, where specificmethods for producing a number of different hydrogels which are suitablefor use in the subject can be found in the Experimental Section infra.Alternatively, suitable hydrogels or precursors thereof may be purchasedfrom various commercial sources, where such sources include: Shearwater,BASF, Polysciences and the like.

Specific hydrogels suitable for use in the subject methods are describedin U.S. Pat. Nos.: 5,626,863; 5,573,934; 5,567,435; 5,529,914;5,514,380; 5,476,909; 5,041,292, the disclosures of which are hereinincorporated by reference. Of particular interest in many embodiments ofthe subject invention are the hydrogels described in U.S. Pat. No.5,410,016, the disclosure of which is herein incorporated by interest.Specific hydrogels of interest include: cross-linked poly(ethyleneglycol diacrylate); cross-linked poly(ethylene oxide)-(polypropyleneoxide)-poly(ethylene oxide)diacrylate; and the like.

The blood composition is contacted with the hydrogel under conditionssufficient for at least some of, and in many cases a substantial amountof, at least the water component of the blood composition to be absorbedfrom the blood composition into the hydrogel to produce a fibrinogenrich composition and a swollen hydrogel. By substantial amount of wateris meant at least about 10%, usually at least about 50% and more usuallyat least about 90%-95% of the initial water present in the bloodcomposition. Any convenient means of contacting the blood compositionwith the hydrogel may be employed, such as placing both components intothe same container, mixing or agitating the two components to combinethem, and the like. The amount of hydrogel that is contacted with theblood composition will be sufficient to absorb the desired amount ofwater and other components from the blood composition. Where a dryhydrogel composition is employed, 1 gram of dry hydrogel material willbe used for fluid volume ranging from about 5 to 200 ml, usually fromabout 10 to 150 ml. For a partially hydrated hydrogel composition (wherepartially hydrated means a hydrogel composition comprising from about 20to 50% polymer), one gram of hydrogel will be used for a volume of fluidranging from about 1 to 10 ml, usually from about 2 to 3 ml. The twocomponents will be maintained in contact for sufficient time for thedesired amount of water (as well as other desired components) to beabsorbed by the hydrogel. The amount of time for which contact ismaintained between the hydrogel and the composition will vary dependingon the particular nature of the hydrogel, i.e. composition, degree ofhydration and surface area. For example, a longer period of time may berequired when partially hydrated hydrogels are employed, where a shorterperiod of time may be sufficient when completely dehydrated and/or anexcess of hydrogel is employed. Usually contact will be maintained forat least about 30 min and will generally not be maintained for more than200 hours, where embodiments in which contact does not have to bemaintained for more than about 48 hours are preferred. In the mixture(i.e. absorption mixture) into which the two components are combined inthe contacting step, the pH will be maintained at from 6.5 to 11,usually from about 6.5 to 7.5 and more usually from about 7 to 7.5. Thetemperature of the absorption mixture will be maintained at from about 0to 40, usually from about 0 to 30 and more usually from about 4 to 30°C. c In addition to the blood composition and hydrogel, the absorptionmixture may further comprise one or more additional agents which serve avariety of purposes. Such agents include: anticoagulants, such asheparin, buffering agents, e.g. citrate buffer, HEPES, and the like.

Absorption of water and other components, as described above, by thehydrogel in the absorption mixture results in the production of afibrinogen rich phase and a swollen hydrogel. The resultant volume ofprotein concentrate, e.g. fibrinogen rich phase, will usually be lessthan 0.5, usually less than about 0.25 and more usually less than about0.1 of the initial protein comprising composition.

The subject methods of preparing protein concentrate compositions may beused in conjunction with one or more additional protein concentrationand/or separation techniques, e.g. chromatography, precipitation with aprecipitation agent, and the like, where such methods are known to thoseof skill in the art. In certain embodiments of the subject invention,the process may further comprise the addition of one or more additionalvolumes of water to the protein concentrate following the initialabsorption of water and components from the initial fluid. For example,a first volume of serum could be contacted with a hydrogel and reducedto 0.1 of its initial volume following absorption of water by thehydrogel. A volume of water could then be added to the reduced volumeand absorption be allowed to occur for a second time, whereby furthercomponents are removed from the concentrate. This process could berepeated a plurality of times, as desired.

The fibrinogen rich phase is then removed from the swollen hydrogel toyield the subject fibrinogen rich composition. Removal of the fibrinogenrich phase from the swollen hydrogel may be performed using anyconvenient technique or protocol. For example, the fibrinogen rich maybe removed from the swollen hydrogel by aspirating the fibrinogen richphase from the hydrogel surface. Alternatively, the fibrinogen richphase may be removed from the swollen hydrogel through centrifugation,which separates the two phases. Other techniques that may be employedinclude: decanting, filtration, simple mechanical separation, and thelike.

The above process may be repeated one or more additional times to obtaina final protein rich composition of desired characteristics.

Separation yields a fibrinogen rich composition. The fibrinogen richcomposition, because of the method by which it is produced, will be moreconcentrated in protein than the initial composition, where in certainembodiments the total protein concentration will be about 10 timesgreater, and in many embodiments the total protein concentration will beabout 5 to 9 times greater than the protein concentration of the initialcomposition. The concentration of fibrinogen in the fibrinogen richcomposition will range from about 1 to 100, usually from about 1 to 30and more usually from about 1 to 2 mg/ml, where in those embodiments inwhich the fibrinogen rich composition is to be employed in a fibrin gluesystem, the fibrinogen concentration will range from about 30 to 60mg/ml.

Depending on the particular nature of the hydrogel system employed, theresultant fibrinogen rich compositions may be substantially free of oneor more low molecular weight components naturally present in the initialblood composition, such as albumin, growth factors, and the like. Forexample, where hydrogels with a molecular weight cutoff, as describedabove, of 100,000 daltons are employed, the resultant fibrinogen richcompositions will be substantially free of components having a weightthat does not exceed 100,000 daltons. Of particular interest in certainembodiments is the preparation of fibrinogen rich compositions withreduced albumin concentration, i.e. compositions that have reducedalbumin concentrations over that which would occur during theconcentration process.

The subject fibrinogen rich composition may be used as produced above ormodified through the addition of one or more different agents thatmodulate the chemical and/or physical nature of the composition.Additional agents which may added to the composition include: proteinsassociated with coagulation, e.g. Factor II, fibronectin and the like;viscosity modifiers, such as collagen, sodium hyaluronate, and the like;antioxidants, such as hydroquinone, vitamin E, and the like; bufferingagents, such as HEPES and the like; processing aids, antifibrinolyticagents, platelet activating agents, wound healing agents, and the like.Of particular interest in certain embodiments of the invention,particularly where the composition is to be used in a firbin sealanttissue adhesive, is the modification of the fibrinogen rich compositionto comprise a visualization agent. Visualization agents (i.e. agentsthat may help a surgeon see those tissues to which the fibrin glue hasbeen applied) that find use include blood compatible, non-toxicflourescent compounds and chromogenic dyes, where specific visualizationagents of interest are those that provide for color contrast with thebackground tissue, with blue and green being preferred colors, wherespecific agents include: indocyanine green, FD & C no. 1, FD & C no. 6,eosin, fluorescein, and the like.

Following preparation of the fibrinogen rich composition, thecomposition may be used immediately or stored for use at a subsequenttime. Any suitable storage means may be employed, where the storagemeans will typically be sterile where the composition is to ultimatelybe used in a physiological setting, e.g. where it is to be used in adrug delivery vehicle, as a surgical adhesive and the like, as furtherdescribed below. One convenient means of storing the composition is tolyophilize the composition and package the lyophilized product in asterile packaging for subsequent use, such as a syringe. Alternatively,the composition may be stored at a reduced temperature, e.g. from about4 to −20° C. or lower.

The subject method of preparing the fibrinogen rich composition may beperformed at both the laboratory scale and scaled up to produce largeamounts of fibrinogen rich composition, e.g. from pooled plasma. On thelaboratory scale, the process of the invention may be conducted usingstandard laboratory and medical equipment. For example, whole blood maybe withdrawn from a mammalian host into a syringe containing ananticoagulant and emptied into a sterile centrifuge tube containing theabsorbable hydrogel, e.g. hydrogel beads. The hydrogel is then allowedto swell in the presence of whole blood for a period of time sufficientfor the desired amount of protein concentration to occur, as describedabove. The resultant concentrated plasma, i.e. fibrinogen rich phase, isthen separated from swollen hydrogel and other cellular material, e.g.red blood cells, by any convenient means, e.g. centrifugation andaspiration using a blunt needle. Such laboratory scale processes findparticular use in situations where one wishes to prepare an autologoustissue adhesive, e.g. where the adhesive is prepared from a patient'sown blood prior to, or even during, a surgical operation. For scale-uppreparation from large volumes of initial blood composition, pooledblood plasma, which may be screened for viruses such as Hepatitis B, andAIDS, may be transferred to a batch reactor containing sterileabsorbable hydrogel beads, e.g. photo polymerized polyethylene glycoldiacrylate (molecular weight range 20,000 daltons), and the like. Thebeads may then be allowed to absorb water present in the pooled plasmauntil a desired level of fibrinogen concentration is reached. Afterreaching the fibrinogen level (typically>30 mg/ml), the beads areseparated from the concentrated solution, i.e. fibrinogen richcomposition, either by filtration, centrifugation, or through theirsettling out due to gravity.

The subject fibrinogen rich compositions find use in a variety ofapplications, as components of tissue adhesives, in drug deliveryvehicles, hemostatic agents, wound healing agents, and the like. Intissue adhesives, the subject compositions will be used in combinationwith a coagulation component, usually a solution of thrombin and calciumions, where upon combination of the composition with the coagulationcomponent a fibrin sealant composition is produced which sets into abiocompatible proteinaceous matrix, i.e. a fibrin gel or sealant. Avariety of different methodologies and devices for performing suchmethodologies have been developed for preparing fibrin sealants fromfibrinogen rich compositions, and such methodologies and techniques aresuitable to prepare a fibrin sealant from the subject compositions. Aswith the preparation of fibrin sealants from fibrinogen compositionsprepared from prior art methods, fibrin sealants may be prepared fromthe subject compositions by applying them to a tissue repair site (i.e.a tissue site to which adherence of a second tissue is desired) eithersimultaneously or sequentially with a thrombin/calcium ion settingcomposition, where suitable thrombin/calcium compositions are readilyavailable from commercial sources and known to those of skill in theart. To apply the fibrin sealant, the two components described above maysimply be applied sequentially or simultaneously to the tissue repairsite via a needle or syringe or other application system. In certainembodiments, it is preferred to apply the components sequentially so asto “prime” the tissue, which results in improved tissue adhesiveresults. Where the tissue is primed, a first component of the fibringlue, e.g. the crosslinker or thrombin, is applied to the tissue repairsite. Next, the fibrinogen components, which may include additionalcrosslinker/thrombin, is applied. Instead of manually applying thefibrin glue to the tissue repair site, one may use specialized devicesfor applying the two components of the fibrin glue. Representativedevices which may be used include those described in U.S. Pat. Nos.4,874,368; U.S. Pat. No. 4,631,055; U.S. Pat. No. 4,735,616; U.S. Pat.No. 4,359,049; U.S. Pat. No. 4,978,336; U.S. Pat. No. 5,116,315; U.S.Pat. No. 4,902,281; U.S. Pat. No. 4,932,942; WO 91/09641, and Tange, R.A., Fibrin Sealant in Operative Medicine: Otolaryngology-Vol. 1 (1986),the disclosures of which are herein incorporated by reference.

Fibrin sealants as prepared above with subject compositions find use astissue adhesives in a variety of clinical applications, where suchapplications are reviewed in Schlag & Redl, Fibrin Sealant in OperativeSurgery (1986) Vol. 1-7, and include: cardiovascular surgery,orthopaedic surgery, neurosurgery, ophthalmic surgery, general surgeryand traumatology, plastic reconstruction and maxillofacial surgery,otorhinolaryngology, and the like. Where convenient, the fibrin sealantmay comprise a visualization agent (e.g. where the sealant is used in alaproscopic method). The visualization agent, including those describedabove, may be present in one or both of, but usually one of, thecomponents of the fibrin sealant, i.e. it may be present in thefibrinogen rich component (where it may have been introduced to thefibrinogen rich composition after its production or introduced into theinitial fluid from which the fibrinogen rich compositions is produced(with the amount used selected in view of the absorption of agent by thehydrogel)) and/or the thrombin/calcium ion component.

The subject fibrinogen rich compositions according to the subjectinvention may also be used in a fibrin gel or sealant or matrix forbiologically active agent or drug delivery. Active agents of interestwhich may be delivered with fibrin sealant compositions prepared asdescribed above include: proteins, carbohydrates, nucleic acids, andinorganic and organic biologically active molecules, where specificbiologically active agents include but are not limited to: enzymes,antibiotics, antineoplastic agents, local anesthetics, hormones,antiangiogenic agents, antibodies, neurotransmitters, psychoactivedrugs, drugs affecting reproductive organs, and oligoncucleotides suchas antisense oligonucleotides. To prepare fibrin sealant drug deliverycompositions with the subject fibrinogen rich compositions, one maysimply combine a therapeutically effective amount of the active agentwith one or both of the components of the fibrin sealant and prepare thesealant as described above.

In a preferred embodiment of the subject invention, the active agent oragents are present in a separate phase from the fibrin gel whichprotects the fibrin gel while it is setting from adverse effects of theactive agent and/or modulates the release kinetics of the active agentfrom the gel, where “separate phase” could be: oil (oil-in-wateremulsion); biodegradable vehicle; and the like. Biodegradable vehiclesin which the active agent may be present include: encapsulationvehicles, such as microparticles, microspheres, microbeads, micropelletsand the like, where the active agent is encapsulated in a bioerodible orbiodegradable polymer such as: polyanhydride, polyglycolic acid,polylactic acid, polyorthocarbonate, polycaprolactone, polytrimethylenecarbonate or their copolymers; caging or entrapping molecules, such ascyclodextrins and the like, etc. Biodegradeable vehicle protected activeagents are particularly preferred where the active agent is anantibiotic, such as gentamycin, tetracylcine, and the like.

In using fibrin sealants prepared from the subject fibrinogen richcompositions as drug delivery vehicles, the fibrin sealant comprisingthe active agent, where the agent may be originally present in thefibrinogen and/or thrombin component of the sealant gel, which isoptionally and preferably present in a biodegradable vehicle, will beadministered to a host prior to setting of the fibrin sealant or gel,where upon administration the gel will set and act as a depot forrelease of the active agent to the host. Such methods of drug deliveryfind use in both systemic and local administration of an active agent.In using the fibrin sealants for drug delivery as described above, theamount of fibrin sealant and dosage of agent introduced into the hostwill necessarily depend upon the particular drug and condition beingtreated. Administration may be by any convenient means, such as syringe,cannula, trochar, and the like.

While various illustrative uses of the fibrinogen rich compositionsprepared by the subject methods have been reviewed above, as explainedsupra the subject methods are not limited to the preparation offibrinogen rich compositions, but can be used to produce other proteinconcentrates. For example, by selecting the appropriate hydrogel withthe appropriate absorptive properties, one can prepare albumin richcompositions.

Albumin rich compositions can be used in the preparation of albumintissue adhesives or glues analogous to fibrin glues. For albumin glues,the albumin present in the albumin rich phase can be crosslinked using anumber of different cross-linking means, including the use of chemicalcrosslinking agents, such as polyaldehydes, thermal crosslinking agents,such indocyanine green in combination with light (e.g. laser emitting at780 nm), and the like. See U.S. Pat. No. 5,583,114, the disclosure ofwhich is herein incorporated by reference.

In certain embodiments of the invention, a water soluble, biodegradeablesynthetic cross-linkers will find use in the preparation of proteingels, such as fibrin glues and albumin glues mentioned above, or othercompositions in which polymeric compounds are cross-linked, as describedherein and in other applications known to those of skill in the art. Thewater soluble, biodegradeable synthetic cross-linkers will bemulti-functional, where by multifunctional is meant that thecross-linkers will be other than monofunctional, where illustrativemultifunctional cross-linkers are difunctional, trifunctional,tetrafunctional, pentafunctional, hexafunctional . . . up to“n”-functional, where n is an integer representing the number ofdifferent end cap functional regions present on the polymericcrosslinkers.

The water soluble, biodegradable polymeric crosslinkers will have a corethat is a biologically inert polymeric unit; (b) an extension at eachend of the core that is a biodegradable polymeric unit; and (c) an endcap on each extension that is a reactive moiety.

The biologically inert polymeric unit or region that makes up the coreof the compound will be an inert polymeric block that is biocompatible,where the block may or may not be biodegradable. Preferably, the core isa polymeric region that is water soluble, where preferred polymers fromwhich the core may be derived include: polyethers, e.g.polyakyleneoxides such as poly(ethylene glycol), poly(ethylene oxide),poly(ethylene oxide)-co-(poly(propyleneoxide)block copolymers;poly(vinyl alcohol); poly(vinyl pyrrolidone), poly(amino acids),poly(ethyloxzoline), dextran and the like, where polyethers, and moreparticularly polyoxyalkylenes, are preferred.

The biodegradable extension component of the subject crosslinkers willbe a component that degrades under physiological conditions intonon-toxic products, where the biodegradable extension component willgenerally be hydrolyzable. Hydrolyzable components of interest include:polymers, copolymers and oligomers of: glycolide, dl-lacide, d-lactide,l-lactide, caprolactone, dioxanone and trimethylene carbonate or theircopolymers and the like. In many instances it is desirable to havepolylinkers with enzymatically hydrolyzable biodegradable extensioncomponents, e.g. regions cleavable by metalloproteinases andcollagenases, where such components will generally be peptidic.Illustrative biodegradable regions include: polyhydroxyacids,polyorthocarbonates, polyanhydrides, polylactones, polyaminoacids andpolyphosphates and the like. The size or length of biodegradable regionmay be varied to in a number of ways. For example, by using acrosslinker based on glycolide in the crosslinking of proteins, theresultant crosslinked protein degrades much faster than crosslinkedproteins based on polycaprolactone cross-linkers. Thus, by choosingappropriate biodegradable polymer regions in the crosslinker, a suitabledegradation profile of the resultant crosslinked protein can beobtained. With difunctional cross-linkers, peptidic biodegradableregions are preferred.

The reactive moiety that is the end-cap on each of the extensions is anactivated functional group which provides for covalent bonding toproteins under in vivo conditions without free radical initiation. Suchgroups include carbodiimidazole, sulfonyl chloride, chlorocarbonates,n-hydroxysuccinimdyl ester, succinimidyl ester, epoxides, aryl halides,sulfasuccinimidyl esters, maleimides, and the like.

FIG. 5 provides further description of such polymeric cross-linkers. Thebiodegradable region of the crosslinker is represented by () theactivated reactive end-group is represented by (); and the inert,biocompatible block is represented by (). Structure A shows a linearwater soluble biodegradable polymer end-capped with two reactivefunctionalities like carbodiimida zole (CDI). The linear water solubleregion is a polyalkylene oxide, preferably polyethylene glycol, which isextended with the biodegradable region which is a copolymer orhomopolymer of trimethylene carbonate. This polymer is then terminatedwith CDI. Structure B is a branched or star shaped trifunctionalbiodegradable polymer which has inert polymer at the center. The inertpolymer is extended with oligomeric biodegradable extensions which arethen terminated by reactive functional end group. Structures C and Dshow a multibranched tetrafunctional biodegradable polymer. This polymeragain has a water soluble core at the center which is extended by smalloligomeric extensions of biodegradable polymer and then terminated withreactive functional groups like carbodiimidazole groups. Structure Eshows a multifunctional star or graft type biodegradable polymer. Thispolymer has a water soluble polymer, like polyalkylene oxide, at thecore which is then extended with biodegradable polymer. Thebiodegradable polymer is terminated with reactive end groups.

The structures shown in FIG. 5 have three common structural features. Awater soluble core which is made by a polymer like polyethylene glycol.The core is extended with an oligomeric extension of biodegradablepolymer such as polylactic acid. The degree of polymerization ofbiodegradable core is kept small (preferable less than 10) to maintainwater solubility. The molecular weight of PEG is kept less than 35,000daltons, as higher molecular weights may be difficult to eliminate bythe body. The biodegradable polymer is then end-capped with reactivefunctional groups which are capable of reacting with proteins or itsfunctional groups like amine, thiols and carboxylic acids preferably inwater and preferably at pH 4 to 9, more preferably at pH 7.0 to 7.4.

The polymeric crosslinkers may be prepared using variety of differentsynthetic methods. In a preferred embodiment, the polymer described instructure A can be obtained by a ring opening polymerization oftrimethylene carbonate initiated by a dihydroxy compound such aspolyethylene glycol molecular weight 2000 d in presence of a suitablecatalyst such as stannous octoate. The hydroxy groups of the copolymerthus obtained are then activated with carbodiimidazole (CDI). The CDIactivated polymer can then be reacted with a protein concentrate asprepared by this invention to form a crosslinked gel. The reactionconditions of the crosslinking reaction will depend on the nature ofactivating group employed. Preferred reactions are at pH 5 to 8, mostpreferred at pH 7.4. The resultant gel degrades due to hydrolysis of thebiodegradable polymer such as polytrimethylene carbonale polymer insidethe crosslinker. The crosslinking density of the resultant network canbe controlled by the overall molecular weight of the structure. A lowermolecular weight such as 600 will give much higher crosslinking densityas compared to a higher molecular weight crosslinker such a withmolecular weight 10000 daltons. The high molecular weight linearcrosslinker is preferred in obtaining elastic gels. The reaction betweenproteins and crosslinkers can be carried out directly on tissues andused as tissue glue. A trifunctional biodegradable crosslinker can beobtained by initiating a polymerization of lactide with trihydroxypolyethylene glycol (ethoxylated trimethylol propane triol) in presenceof stannous octoate. The degree of polymerization of lactide is keptless than 5. This is achieved by choosing a molar ratio of PEG withlactide (molar ratio of lactide to PEG is 6,2 per branch). ThePEG-lactate trifunctional polymer is isolated. The hydroxy end groups ofcopolymers are then activated with CDI. Since this a trifunctionalcrosslinker, it will give higher crosslinking density as compared tosimilar molecular weight difunctional crosslinker. This gives additionalflexibility in controlling crosslinking density of a crosslinkedstructure and hence their mechanical and biodegradation properties. Thetertafuctional structures are obtained by reacting the polyalkyleneoxide copolymer such as TETRONIC 908 (obtained from BASF corporation)with caprolactorie in presence of stannous octoate. The reaction iscarried out in melt at 180° C. for 6 hours under nitrogen atmosphere.The molar ratio of caprolactone to TETRONIC 908 is kept to 12, whichmaintains water solubility of TETRONIC 908-caprolactone copolymer inwater. The polymer is activated with CDI and used in crosslinkingreaction with proteins.

The protein concentrates prepared by the subject methods also find usein the preparation of novel hydrogel wound dressings. To preparehydrogel wound dressings from the subject protein concentrates, such asthe fibrinogen rich compositions discussed above, where the proteinconcentrate serves as the “aqueous composition component,” the proteinconcentrates are combined with a macromonomer component and across-linking agent under conditions sufficient to produce a hydrogelcomprising the protein concentrate composition interspersed throughoutthe cross-linked gel network.

Macromonomers that find use in the preparation of hydrogel wounddressings are non-toxic, water soluble, non-ionic macromonomers thatcomprise a polymerizable group, preferably an addition polymerizablegroup, where macromonomers which can be crosslinked via free radicalpolymerization are preferred. For example, water soluble polymers havingunsaturated polymerizable groups such as acrylate, methacrylate,itaconate, and the like find use. Water soluble polymers of interestwhich can be modified with unsaturated polymerizable groups include:polyethers, such as polyalkyleneoxide polymers and copolymers,polyethyleneoxide, polyethylene glycol,polyethyleneoxide-polypropyleneoxide block, random or graft copolymers;polyvinyl alcohol; polyvinyl pyrrolidinone; and the like, wherepolyether polymers or derivatives thereof are particularly preferred asmacromonomers. If desired, two or more macromonomers can becopolymerized to obtain suitable hydrogel properties.

While preparation of the hydrogel wound dressings of the subjectinvention has been described in terms of using the protein concentratesof the present invention as the aqueous composition component that iscombined with the macromonomer and the cross-linking agent, otheraqueous composition components may also be employed to prepare hydrogelwound dressings according to the subject invention. Other aqueouscompositions that may be employed as the aqueous composition componentto prepare the subject hydrogel wound dressings include: whole blood,serum, platelet rich plasma (where the platelets may or may not beactivated), tissue culture medium, and the like.

The hydrogel wound dressings of the subject invention can be preparedjust prior to use or prepared and then stored for subsequent use.

The cross-linking agent is generally a non-toxic polymerizationinitiator, such as a free radical initiator. Several free radicalinitiating systems can be used to polymerize the macromonomerscontaining polymerizable groups such as acrylate or methacrylate. Someof the preferred examples are: Darocur 2959 (initiated around 360 nm),Irgacure 651 (initiated around 360 nm), eosin-triethanol amine(initiated around 510 nm), methylene blue-triethanol amine (initiatedaround 632 nm), sodium persulfate (initiated around 50° C.), ammoniumpersulfate (initiated at 50° C.), Glucose oxidase-glucose-ferroussulfate (initiated around 37° C. in presence of dissolved oxygen in theformulation) and the like.

Optionally, other pharmaceutically acceptable catalysts and cocatylystscan be added to the macromonomer solution to accelerate thepolymerization speed and/or to improve its shelf life. For example,small amounts of vinyl pyrrolidinone (concentration around 1-10micrometers per ml) can be added while using eosin-triethanolphotoinitiating system. Inhibitors such as hydroquinone may be added toprevent premature polymerization of macromonomer during its storage.Optionally, the wound dressings described may be prepared with bioactivecompounds such as antibiotics to reduce the bacterial infection.

Other agents that may be included in the composition to modulate theultimate properties of the hydrogel wound dressing prepared therefrominclude proteinaceous polymers, such as collagen, other polymericcompounds, such as hyaluronic acid (including derivatives thereof),dextran and the like. Of particular interest are hydrogel wound dressingcomposites which include, in addition to the hydrogel, at least one ofcollagen and hyaluronic acid, where in such composites, PEG is thepreferred hydrogel.

Hydrogel wound dressings produced in accordance with the presentinvention can be prepared with desired physical and chemical propertiesby choosing specific structural features, such as nature ofpolymerizable group of the macromonomer, the number of polymerizablegroups present per macromonomer chain, the chain length and chemicalstructure of the macromonomer, where the modulation of such parametersto obtain a hydrogel wound dressing of desired characteristics is withinthe skill of those in the art.

The hydrogel wound dressing composite produced according to the presentinvention can be produced in various shapes and sizes, such as: films,ropes, rods, plugs, thin or thick sheets, moldings and laminates.Hydrogel wound dressings prepared according to the present invention canbe modified further, if necessary or desired, by the addition ofpharmaceutically acceptable antioxidants, plasticizers, coloring agents,fillers, fibers, fiber meshes, adhesive backing sheets and the like.

Optionally, the hydrogel wound dressing composite produced according tothe present invention can be reinforced with flexible or rigid fibers,fiber mesh, fiber cloth and the like to produce a fiber reinforcedhydrogel wound dressing. The insertion of fibers or fibrous structuresimproves flexibility and tear resistance of the hydrogel wounddressings. Such structures can be produced using any convenientprotocol. In a preferred method, the aqueous macromonomer formulation isadded to the fiber cloth or net such as cotton gauze. The liquid thenflows into the interstices of cloth and is then polymerized to produce ahydrogel, where care is taken to ensure that the fibers or fiber meshare buried completely inside the hydrogel material. The fibers used inreinforcing the hydrogel network are preferably hydrophilic in nature toensure better compatibility with the hydrogel network. Also atransparent, flexible plastic film such a polyethylene plastic filmwhich is permeable to oxygen, may be applied on top of the hydrogelwound dressings (opposite side of wound contact) and which may suppliedwith a plastic mold described in this invention. This plastic filmprevents the moisture loss from the hydrogel.

The hydrogel wound dressings produced according to the present inventioncan be used on variety of wounds. The wounds may be surgical wounds,first, second or third degree bums, skin lesions, decubitus ulcers,venous ulcers, bed sore and the like.

Also provided by the subject invention are kits for preparing thesubject fibrinogen rich compositions, kits for preparing the wounddressings and kits for preparing fibrin sealants from the subjectfibrinogen rich compositions. Kits for preparing the fibrinogen richcompositions will comprise at least a hydrogel composition andinstructions for preparing the fibrinogen rich composition according tothe methods of the subject invention, where the instructions may bepresent in the kit as an insert, incorporated into the containers and/orpackaging of the kit, and the like. Kits for preparing the fibrinogenrich compositions may further comprise anticoagulants, e.g. heparin, andone or more containers, e.g. syringes, vials and the like, for use inpreparing the fibrinogen rich composition, and the like; where the kitcomponents will generally be sterile, particularly where the ultimateuse of the composition involves introduction into a host, e.g. a patientundergoing surgery.

Kits for preparing a fibrin sealant according to the subject inventionwill comprise at least a fibrinogen rich composition prepared accordingto the subject invention and a thrombin/calcium setting componentpresent in separate containers. The kit may further comprise a devicefor delivery the fibrin sealant to a tissue repair site, as describedabove. Optionally, the kits may also comprise a visualization agentand/or an active agent, preferably present in a biodegradable vehicle.As with the kits for preparing the subject fibrinogen rich compositions,kits for preparing fibrin sealants will also typically compriseinstructions for preparing the fibrin sealant, which instructions may bepresent as a package insert and/or associated with the containerspresent in the kit and/or packaging of the kit.

Kits for preparing the subject wound dressings will comprise at leastthe macromonomer component described above and instructions for carryingout the subject methods of preparing the wound dressings. Generally, thekits will also comprise a crosslinking agent. The kits may furthercomprise a fluid of interest which is to be entrapped in the hydrogel,such as the concentrated protein composition or other fluids, asdescribed above. Other components that may present in the kits include:a container/mold for preparing the wound dressings, and the like.

Also provided are devices for automatically performing one or more ofthe steps of the subject method to prepare a protein concentrate from aninitial protein comprising aqueous composition. The devices of subjectinvention will generally comprise a container means having a quantity ofhydrogel and of sufficient volume for the hydrogel to come into contactwith the initial protein composition. The container will furthercomprise at least one opening for introducing the initial compositioninto the container and removing the resultant protein concentrate fromthe container. Generally, the devices will comprise a contacting meansfor ensuring fluid transfer between the fluid introduced into thecontainer and the hydrogel present therein. In some embodiments, thecontacting means is a simple as a polymeric interface layer that isresistant to protein absorption/adsorption. In other embodiments, thecontacting means may comprise a filtering means, such as a glass filteror membrane, which is impermeable to the hydrogel. Representativedevices according to the subject invention are further described interms of the figures.

In FIG. 6 is represented a first device embodiment according to thesubject invention. Device 10 is a plastic/glass bottle 12 (could also bea bag) containing hydrogel beads 14 separated from the remainder of thebottle interior by filter mesh 18 which is permeable to liquids but notto the hydrogel beads (i.e. the contacting means). The bottle has anarrow neck 20 that has a volume measurement printed thereon 22. Inusing this device embodiment, plasma is added to the hydrogel beads andthe swelling process is started. The bottle is inverted every 10 minutesto separate the concentrated (the filter prevents the hydrogel fromentering the narrow neck region. In the narrow neck region (uponinversion), the volume of plasma is measured. When the desired volume isreached, the concentrated plasma is removed using a suitable technique.

FIG. 7 depicts a second embodiment of a device according to the subjectinvention. Cylindrical device 30 has entry port 32 for the introductionof fluids into the device. The entry port has a seal (e.g. silicon suchas those employed on blood banking blood bags) through which fluids canbe sterily introduced into the device or a removable lid (not shown)which can be used in an asceptic technique. Areas 34 contain hydrogelwhich can absorb fluid and smaller molecules. For use in preparingcryoprecipitate, the hydrogel might have a molecular weight cutoff ofabout 80,000 so albumin would be absorbed into the hydrogel. In theembodiment shown, the hydrogel compartment would not change physicalvolume, but has room for the hydrogel to swell and expand. However,other embodiments in which the physical volume of the hydrogelcompartment is expandable are envisioned. Collection compartment 36 is acylindrical shaped cup (though other shaped compartments are alsocontemplated, such as rectangular etc.) that has walls made of materialthat does not absorb protein and is also impermeable to fluid. Exit port38 allows for the sterile removal of fluid, i.e. a septum that can bepunctured with a needle.39 is the top layer of the hydrogel compartmentwhich is a material resistant to protein adsorption/absorption (i.e. thecontacting means). The top layer could be fabricated from an appropriatepolymeric material, e.g. PEG, or be a membrane which allows for passageof water and other small molecules, such as those used in thepurification industry. Use of a membrane provides greater flexibility inthe nature of the hydrogel that may be employed, as direct contact ofthe protein components of the fluid and the hydrogel can be prevented bythe membrane. Accordingly, the hydrogel in such devices need notnecessarily be a hydrogel that is non-denaturing, as that term isemployed above. FIG. 7(e) provides a top view of the device. FIGS. 7(b)to 7(d) show the device in use. In using the device to prepare a proteinconcentrate from an aqueous composition, fluid 40 is introduced throughport 32. Water and other small molecules are absorbed into hydrogel 34.As the fluid is absorbed, the level of the fluid falls until it reachesthe top of the collection compartment 36. Concentrate is then removedfrom the collection compartment through exit 38. The device isconfigured with dimensions chosen based on the intended nature of use.For example, in the preparation of a fibrinogen rich composition fromplasma, in many situations the device will have dimensions sufficient tohold up to 1 unit of blood. Where desired, the entire device can berotated at low rpm (usually less than 500) to facilitate mixing of thefluid and reduction of fouling by proteins of the top or interface layerof the hydrogel or the membrane 39 covering the hydrogel component.Furthermore, coatings or active coatings or gel microspheres with activecoatings could be placed on the periphery of the device (on the insideof the outside wall of the device above the hydrogel compartment) whichwould specifically absorb albumin, other proteins or specific cell typesfor cell purification. If the membrane 39 over the fluid absorbingcomponent 34 is properly selected, a wider range of swelling polymersmay be employed as the hydrogel component as mentioned above.Accordingly, polymer compositions such as those found in disposablediapers to absorb urine may be employed. Alternatively, the device couldoperate with hydrogel beads that are excluded from the collection cup 36by having a means over the collection cup.

The following examples are offered by way of illustration and not by wayof limitation.

Experimental

A. Materials and Methods

Polyethylene glycols (Merck, mol. wt 20000 and BDH mel. wt 6000) wereused as received. PLURONIC® and TETRONIC® polyols were purchased fromBASF corporation. Photoinitiator DAROCURE® 2959 was purchased from CibaGeigy. The biodegradable polymers like polylactic acid, polyglycolicacid are purchased from Polysciences. All other reagents, solvents areof reagent grade and are purchased from commercial sources such asFluka, Aldrich and Sigma. Small laboratory equipment was purchased fromFisher or Cole-Parmer.

B. Synthesis of Hydrogels

1. Synthesis of Polyethylene Glycol Diacrylate

In a 1,000 ml 3 neck reaction flask, attached with condenser, nitrogeninlet and thermometer, 100 grams of PEG (molecular weight 20,000daltons) and 600 ml dry toluene are added. After distilling 100 ml oftoluene from the flask, the PEG solution is cooled to 50-60° C. To thismixture, 2.1 ml of triethylamine and 1.2 ml of acryloyl chloride isadded under nitrogen atmosphere. The reaction is stirred for 10-12 hoursat ambient temperature under nitrogen atmosphere. The triethylaminehydrochloride (a reaction byproduct) is removed by filtration and theproduct is recovered by pouring the filtrate in to large excess hexane.It is further purified by several dissolution precipitation steps usingtetrahydrofuran (THF)-hexane as solvent-nonsolvent respectively. Theproduct is further dried at 40° C. under vacuum until constant weight isobserved. Yield 60 gram.

Other macromonomer containing PEG with different molecular weights, anddifferent number of unsaturated groups are easily synthesized using theprocedure given above. For example, 30 grams PEG molecular weight 6000daltons is reacted with 2.67 g acryloyl chloride and 3.03 g triethylamine using a similar procedure mentioned above to give PEG 6,000diacrylate. Similarly PEG 10000 diacrylate is prepared by reacting 30grams of PEG molecular weight 10,000 Da with 1.82 g of triethyl amineand 1.61 g of acryloyl chloride. Polyethyleneoxide (PEO) molecularweight 35,000 Da (30 g) is reacted with 0.52 g of triethyl amine and0.46 g of acryloyl chloride to get a corresponding diacrylatederivative. Some polyethylene glycol based macromonomers may also bepurchased using commercial sources such as Sartomer, Polysciences.

2. Synthesis of Polyethyleneoxide-Polypropyleneoxide-polyethyleneoxideDiacrylate

50 grams of PLURONIC® F127 (a PEO-PPO-PEO block copolymer with 70% PEOcanient, molecular weight 12500 daltons, purchased from BASFcorporation) is dried under vacuum at 80-100° C. The polymeric diol isthen transferred to a 3 neck reaction flask equipped with nitrogen inletand thermometer. 500 ml toluene, 3.3 ml of triethylamine amine and 1.9ml of acryloyl chloride are added to the reaction mixture under drynitrogen atmosphere. The reaction mixture is stirred overnight andfiltered to remove triethylamine hydrochloride. The filtrate is added to3000 ml hexane to precipitate the diacrylate derivative of PLURONICF127. The macromonomer is purified by several dissolution-precipitationsteps from THF-hexane solvent-nonsolvent system. Finally the diacrylateis dried under vacuum at 40° C. to a constant weight.

Other derivatives of PLURONICS with different arrangement of PEO-PPOblocks and with different HLB values can also be acrylated in a similarmanner.

3. Preparation of Sterile Gels Using Photopolymerization Method

10 grams of PEG diacrylate, synthesized by a procedure described above,are dissolved in 20 grams of phosphate buffer solution (pH 7.4, 0.2 g/LKC1, 0.2 g/L of KH₂PO₄, 8.0 g/L of NaCl and 1.15 g/L Na₂HPO₄). To thissolution 600 μl of photoinitiator solution (300 mg of Darocur® 2959,Ciba Geigy, dissolved in 700 mg ethanol) is added. The followingprocedure is carried out in a sterile hood. The aqueous macromonomersolution is sterile filtered using 50 ml syringe and 0.2 mm syringefilter. 200 μl of sterile solution is transferred into transparentplastic mold (a single well of a sterile 96 well tissue culture plate).The solution is then exposed to long wavelength UV light (Blak Ray lightsource, model 3-100A, Flood 365 nm, intensity 10 mW/cm²) for 120seconds. The polymerized gel is removed from the mold. The resultant gelmay be freeze dried or freeze dried coupled with limited hydrationfollowing preparation. Several of such hydrogel beads are synthesizedand stored in a sterile container for further use.

4. Preparation of Sterile Gel Microspheres

5 grams of PEG diacrylate, molecular weight 10,000 daltons are dissolvedin 15 grams of trietholamine/triethanolamine hydrochloride solution (90mM, pH=7.4) in a 50 ml amber colored glass bottle. To this solution 30μl of Eosin Y solution (1 mg/ml in PBS) and 30 μl vinyl pyrrolidinoneare added. The mixture is protected from visible light using aluminumfoil. The following operations are carried out in a sterile hood: Themacromonomer solution is filtered using 50 ml syringe and 0.2 μm syringefilter. The filtered sterile solution is filled into 50 ml plasticsyringe (wrapped in aluminum foil for protection against light) with 22gauge needle. A 100 ml of sterile mineral oil which is constantly beingstirred using a magnetic stir bar is exposed to green light source(American Argon ion laser, Model 905 emitting at 532 nm, 100 mW/cm²).The macromonomer solution is flushed out of needle and the droplets arecollected into sterile mineral oil which is under green lightirradiation. After 5 minutes of irradiation of monomer solution to greenlight, the photopolymerized hydrogel beads are separated from mineraloil by filtration. The hydrogel beads are further washed with sterilehexane to remove traces of mineral oil. The hydrogels beads are thenstored in sterile saline solution for 5 hours to remove initiatorfragments, unreacted or uncrosslinked macromonomer etc. The washed beadsare then isolated by filtration and lyophilized under sterileconditions.

5. Synthesis of Multifunctional Thermosensitive Macromonomer

30g of TETRONIC 908 polyol is dissolved in 400 ml dry benzene. 100 ml ofbenzene is distilled to remove traces of water from the polyol. Thesolution is cooled to 30° C. and 1.45 g triethylamine and 1.90 gacryloyl chloride are added. The reaction mixture is refluxed for 1 hunder argon atmosphere. It is then cooled and then filtered to removedtriethylamine hydrochloride. The filtrate is then added to 2000 mlhexane to precipitate the polymer. The polymer is purified by severalprecipitations from THF-hexane solvent-nonsolvent system. Furthersolvent removal/drying is achieved by vacuum drying overnight at 600° C.

6. Preparation of Hydrogels by Radical Thermal Polymerization Method

10 grams of PEG diacrylate molecular weight 20,000 daltons are dissolvedin 20 gram of PBS buffer (pH=7.4). To this solution 300 mg ammoniumpersulfate are added as a thermal free radical polymerization catalystand the mixture is sterile filtered. This sterile solution is thentransferred into several sterile 5-10 mm diameter glass test tubes. Thetest tubes are then capped and transferred into lab oven maintained at70° C. After 15 minutes, the test tubes are removed, cooled and thepolymerized gels are removed by breaking the glass container. The gelrods are cut into small 1-2 cm pieces and stored. Sterility of gel rodsis maintained throughout the entire operation. The resultant hydrogels,when contacted with water, absorb water over time and increase in mass,as shown in FIG. 3.

C. Synthesis of Drug Compositions

1. Preparation of Rifampin Sulfate Loaded Polylactic Acid Particles

In a 50 ml glass beaker, 2 g of polylactic acid is dissolved in 20 mlmethylene chloride. To this mixture is added 1 g of gentamycin sulfate.The resultant slurry is thoroughly mixed and poured on a glass plate.After initial methylene chloride removal from the slurry, the partiallydried film is dried in vacuum oven at 40° C. for 48 hours. The resultantgentamycin-PLA composite is pulverized into small particles by cryogenicgrinding at liquid nitrogen temperature. The particles may be sieved toobtain a particular size distribution.

2. Preparation of Gentamycin Loaded Microspheres ofPolylactate-co-polyglycolate (50:50) Polymer

0.1 g of Rifampin is dissolved in 5 ml PBS (pH 7.2) containing 10 mg ofbovine serum albumin. The resultant aqueous solution is added to 30 mlmethylene chloride containing 5 grams of polymer and then emulsified bya brief sonification for 30 seconds. The water-in-oil (W/O) emulsion isreemulsified in 2,000 ml 0.1% (w/v) polyvinyl alcohol (PVA) withstirring for 3 hours at room temperature. The hardened microspheres arewashed three times with PBS buffer to remove unencapsulated rifampin.The microspheres are recovered by centrifugation and freeze-drying. Theresidual solvent is removed by drying the microspheres in vacuum oven at37° C. for 48 hours. Alternatively 0.1 g of Rifampin is dissolved in 5ml acetone. The acetone solution is then added to 30 ml solution ofpolylactate-co-polylactate (50:50) in methylene chloride (4% wt/v). Themixture is then spray dried using standard laboratory spray dried.

D. Preparation of Fibrinogen Composition Using Hydrogels

1. In a sterile glass bottle, having a rubber septum cap and containingheparin anticoagulant, 20 ml of patient's blood is transferred using asterile needle transfer technique or standard Schlenk line techniques.The tube is then centrifuged at 3,200 rpm for 10 minutes. The resultantblood plasma is transferred using a sterile needle transfer technique toanother sterile bottle containing 4 g of sterile PEG 20,000 diacrylategel rods prepared by the method described in example 6 above. Thehydrogel selectively absorbs water and other low molecular weightproteins such as albumin, plasminogen and compounds like heparin leavingbehind a concentrated solution of fibrinogen and Factor XIII. Thehydrogel absorption time (usually minutes to several hours) iscontrolled so as to obtain a desired volume/concentration of a finalsolution (typically 90-95% water is removed from the plasma). Thisconcentrated protein solution gels with commercially available thrombin,Ca⁺² ion solution.2. Preparation with Hydrogel Beads

Blood plasma is concentrated with hydrogel beads using a methodanalogous to that described in example D1, above. The process is shownschematically in FIG. 1. In the schematic shown in FIG. 1, dry orpartially dry hydrogel beads are placed in a sterile tube as shown inFIG. 1A. Next, a blood plasma solution is transferred to the tube asshown in FIG. B. The mixture is allowed to remain in contact for asufficient period of time for the hydrogel beads to selectively absorbwater from the plasma, whereby a fibrinogen rich composition isproduced, as shown in FIG. 1C. A small amount of plasma is taken outperiodically and is analyzed for total protein concentration using abiurette method, the results of which are shown in FIG. 4.

E. Preparation of Colored Fibrin Adhesive Solution

1. Indocyanine Green

5 ml of concentrated solution of fibrinogen solution as prepared abovein Example D is added to a sterile bottle containing 0.1 ml indocyaninegreen solution in PBS (conc. 0.1 to 10 mg/ml). The green precursorsolution is easy to visualize and apply under normal and laparoscopicsurgical environment. This green solution is used with thrombin solutionas a colored fibrin adhesive system.

2. Preparation of Fluorescent Adhesive Formulation

5 ml of concentrated solution of fibrinogen solution is added to asterile bottle containing 0.1 ml sodium fluorescine in solution in PBS(conc. 0.1 to 10 mg/ml). The light green fluorescent precursor solutionis easy to visualize and apply under normal and laparoscopic surgicalenvironment. This green solution is used with thrombin solution as acolored fibrin adhesive system.

F. Preparation of Fibrin-biodegradable Microparticle Composite

5 ml concentrated solution of fibrinogen or the commercial fibrinogencontaining precursor of fibrin adhesive is mixed with 0.5 gramspolylactic acid or polyhydroxy acid microspheres loaded with bioactivecompound such as gentamycin. The resultant slurry is transported to alocalized disease site inside the human or animal body using minimallyinvasive surgical device such as laparoscope and crosslinked in situusing thrombin solution.

G. Preparation of Protein Composites Crosslinked with BiodegradablePolymeric Crosslinking Agents

1. Synthesis of Water Soluble Difunctional, Biodegradable CrosslinkerBased on Polyalkylene Oxide

Synthesis of polyethylene glycol-trimethylene carbonate polyol-30 g ofpolyethylene glycol having a molecular weight 2000 is dried at 90-100°C. in a glass sealing tube. The tube is then cooled and transferredinside an air bag where 12.24 g of trimethylene carbonate and 50 mg ofstannous octoate are added to the tube. The glass tube is then sealedunder nitrogen atmosphere and heated with stirring at 155° C. andmaintained at this temperature for 6 hours. The polyethyleneglycol-polytrimethylene carbonate polymer is cooled and recovered bybreaking the glass sealing tube. It is further purified by severalprecipitations from toluene-hexane solvent-nonsolvent system. Theproduct is dried in vacuum at 40° C. and used immediately in theactivation reaction.

2. Synthesis of Water Soluble, Tetrafunctional Crosslinker Based onPolyaikylene Oxide

Synthesis of TETRONIC 908-caprolactone polyol(PCLP)-30 g of TETRONIC 908is charged in a dry 3 neck flask equipped with magnetic stirrer andvacuum inlet. The flask is then heated in a silicone oil bath at 100° C.for 12 hours to dry the TETRONIC 908. The flask is cooled to roomtemperature and 1.642 g of caprolactone and 0.02 g of stannous2-ethylhexanoate are added to the flask. The flask is heated to 180° C.for 6 hours under nitrogen atmosphere. The reaction is product is thendissolved in 200 ml dry toluene (winning of toluene acceleratesdissolution). The toluene solution is added to 2,000 ml dry heptane withconstant stirring. The product is isolated by filtration. Furtherpurification is accomplished by precipitation of toluene solution ofPCLP in heptane. The product is dried in vacuum at 40° C. and usedimmediately in the activation reaction.

3. a) Activation Polyalkyleneoxide Lactate Copolymer withCarbodiimidazole

30 g of TETRONIC 908-caprolactone copolymer is dissolved in 400 ml drybenzene. About 100 ml of benzene is distilled off and the solution iscooled to 50° C. 2.24 g of carbodiimidazole is added to reactionmixture. The mixture is refluxed for 2 h under nitrogen atmosphere. Atthe end of 2 hour period, the solution cooled added to 4,000 ml hexaneto precipitate the polymer. It is further purified by repcated (3 times)precipitation from toluene-hexane system. The polymer is dried undervacuum at 40° C.

b) Activation Polyalkyleneoxide Lactate Copolymer withN-hydroxysuccinimidyl Ester

30 g TETRONIC 908-caprolactone copolymer is dissolved in 400 ml drybenzene. About 100 ml of benzene is distilled off and the solution iscooled to 50° C. 2.50 g of succinic anhydride is added to reactionmixture. The mixture is refluxed for 5 hours under nitrogen atmosphere.At the end of 5 hour period, the solution cooled and then added to 4,000ml hexane to precipitate the polymer. It is further purified by repeated(3 times) precipitation from toluene-liexane system. The polymer isdried under vacuum at 40° C.

c) Activation of Acid Terminated Polymer with Dicyclohexylcarbodiimide(DCC)

10 g of TETRONIC 908-caprolactone succinate prepared by method describedabove is dissolved in 100 ml dry methylene chloride. The mixture iscooled to 0° C. in ice bath and 0.5 g of 4-dimthylaminopyridine and 1 gof dicyclohexylcarbodiimide (DCC) are added. The mixture is stirred at0° C. for 6 hour and filtered. The filtrate is then added to 2,000 mldry hexane to precipitate the activated succinimydyl ester. The productis isolated by filtration, dried under vacuum and stored under argon at4° C.

4. Preparation of Crosslinked Protein Composites

The CDI/succinimydil activated crosslinker polymers as prepared aboveare dissolved in aqueous buffer solution and reacted with albumin richsolutions to form a gel.

H. Preparation of Hydrogel Wound Dressings

1. Preparation of Sterile Hydrogel Wound Dressings UsingPhotopolymerization Method

5 grams of PEG 10,000 diacrylate, synthesized by a procedure describedin example B1, is dissolved in 20 grams of phosphate buffer solution (pH7.4, 0.2 g/L KCl, 0.2 g/L of KH₂PO₄, 8.0 g/L of NaCl and 1.15 g/LNa₂HPO₄). To this solution 100 microliter of a photoinitiator solution(300 mg of Darocur® 2959, Ciba Geigy, dissolved in 700 mg ethanol) isadded. The following procedure is carried out in a sterile hood: Theaqueous macromonomer solution containing photoinitiator is sterilefiltered using 50 ml syringe and 0.2 mm syringe filter. About 10-12 mLof sterile solution is transferred into transparent plastic mold (10cm×5 cm×2 mm). The solution is then exposed to long wavelength UV light(Blak Ray light source, model 3—100A, Flood 365 nm, intensity 30 mW/cm²)for 5 minutes. The polymerized, flexible gel is removed from the mold.This sterile gel can be directly applied over the wound.

2. Gamma Radiation Crosslinking of Macromonomers

3 g of PEG 20,000 diacrylate is dissolved in 12 ml PBS buffer. Theaqueous monomer solutions is sterile filtered into glass mold (cavitysize 10 cm×5 cm×2 mm). The solution is then irradiated at ambienttemperature with a ⁶⁰Co source with doses up to 200 kGy at a dose rateof 1 kGy/h. After irradiation, the sterile crosslinked gel is removedfrom the mold and used as wound dressing.

3. Preparation of Sterile Lyophilized Macromonomer Formulation

3 grams of PEG 10,000 diacrylate, synthesized by a procedure describedin example, is dissolved in 12 grams of phosphate buffer solution (pH7.4, 0.2 g/L KCl, 0.2 g/L of KH₂PO₄, 8.0 gil of NaCl and 1.15 g/LNa₂HPO₄). To this solution 60 microliter of photoinitiator solution (300mg of Darocur® 2959, Ciba Geigy, dissolved in 700 mg ethanol) is added.The aqueous macromonomer solution containing photoinitiator is sterilefiltered using 50 ml syringe and 0.2 mm syringe into a sterile ambercolored 25 ml glass bottle. The solution is lyophilized whilemaintaining sterility to remove water. At the end of the lyophilizationcycle, the vial is capped with a sterile rubber septum. The lyophilizedmacromonomer powder containing photoinitiator is used in making ‘insitu’ formable wound dressings.

4. Autologus or Single Donor Single Donor Blood or Blood ComponentsEncapsulated Wound Dressing

15-20 ml of fresh human or animal blood is withdrawn in a standardsyringe or similar medical device containing anticoagulant such asheparin or acid citrate buffer (2% glucose, 0.15M citrate, pH 4.2, 1 mlbuffer per 10 ml blood). The blood is then centrifuged in order toseparate out the blood plasma. The plasma is then removed from thecentrifuge tube and transferred into a sterile macromonomer lyophilizedpowder containing initiator (such as made in example 3 to make a 5-40%solution, preferably 10% solution of the macromonomer in plasma (someproteins such as fibrinogen may precipitate depending on themacromonomer and its concentration used). The macromonomer-plasmasolution/dispersion is then transferred into sterile plastic dish ormold of size 10 cm×5 cm×2 mm using a sterile syringe and needle. Thecontents of the mold are then exposed to long UV irradiation for 3minutes to crosslink the macromonomer. The crosslinked sterile hydrogelcan be directly applied on the wound.

In another variation of this procedure, 15-20 ml of fresh human oranimal blood is withdrawn in a closed sterile plastic or glass vialwithout anticoagulant. The blood is allowed to coagulate for 30 to 60minutes. The top clear solution (serum) is withdrawn from the vial usinga syringe. This serum is then used in preparation of hydrogel dressingusing a procedure mentioned previously.

5. Preparation of Hydrogel Wound Dressing Containing Platelets

15 to 20 ml of fresh human or animal blood is isolated using a standardprocedure. The blood is then centrifuged at low rpm (around 3,000 rpm)in order to separate the platelet rich plasma. The platelet rich plasmais then added to a sterile lyophilized macromonomer initiatorformulation such as described in example 3. The entire mixture is thenpoured into sterile plastic or glass mold of suitable size for example10 cm×5 cm ×2 mm cavity size and exposed to long UV or visible light for3 minutes to crosslink macromonomer. The gel containing encapsulatedplatelets is removed and may be directly applied on the wound.

Platelets may be activated using thrombin or suitable activating agentbefore or after the encapsulation process. The activated plateletsrelease platelets derived growth factors which are then released incontrolled manner by the hydrogel matrix.

6. Preparation of Hydrogel Wound Dressing Containing Tissue CultureMedium (Without Serum)

A sterile amber colored glass vial containing 3 grams macromonomer andphotoinitiator (similar to described in example 6) is dissolved in 12 mltissue culture medium (for example Dulbecco's modification of Eagle'smedium, a medium suitable for culturing human foreskin fibroblasts). Themacromonomer solution in tissue culture medium is then sterile filteredinto a plastic mold of desired shape for example 10 cm×5 cm×2 mm sizeand exposed to long UV light as mentioned in previous examples. Thepolymerized hydrogel is then removed from the mold and used as wounddressing.

7. Preparation of Hydrogel Wound Dressing Containing Tissue CultureMedium (with Autologus or Single Donor Blood Serum)

15-20 ml of fresh human or animal blood is withdrawn in a standardsyringe and allowed to stand to coagulate for 0.25 to 2 hours. The bloodis then centrifuged in order to separate out the blood serum. 2 ml ofthis blood serum is then mixed with 18 ml of tissue culture medium suchas described in previous example. 12 ml of this mixture is then added toa lyophilized macromonomer formulation prepared as described in example6. This macromonomer is then sterile filtered into a plastic sterilemold 10 cm×5 cm×2 mm size and exposed to long UV light for 3 minutes.The crosslinked macromonomer or hydrogel contains tissue culture mediumand serum.

8. Preparation of Polyalkylene Oxide-hyaluronic Acid Hydrogel WoundDressing

3 grams of PEG diacrylate, synthesized by a procedure described inexample B1, is dissolved in 12 grams of phosphate buffer solution (pH7.2, 0.2 g/L KCl, 0.2 g/L of KH₂PO₄, 8.0 g/L of NaCl and 1.15 g/LNa₂HPQ₄ and 0.5% sodium hyaluronate). To this solution 60 microliter ofphotoinitiator solution (300 mg of Darocur® 2959, Ciba Geigy, dissolvedin 700 mg ethanol) is added. The following procedure is carried out in asterile hood.

The aqueous macromonomer solution containing photoinitiator andhyaluronic acid is sterile filtered using 50 ml syringe and 0.2 μmsyringe filter. About 10-12 mL of sterile solution is transferred intotransparent plastic mold (cavity size 10 cm×5 cm×2 mm). The solution isthen exposed to long wavelength UV light (Blak Ray light source, model3—100A, Flood 365 nm, intensity 30 mW/cm²) for 5 minutes. Thepolymerized, flexible gel is removed from the mold. This sterile gel canbe directly applied over the wound.

9. Preparation of Polyalkylene Oxide-collagen Composite Hydrogel WoundDressing

3 grams of PEG diacrylate, synthesized by a procedure described inexample B1, is dissolved in 12 grams of phosphate buffer solution (pH7.4, 0.2 g/L KCl, 0.2 g/L of KH₂PO₄, 8.0 g/L of NaCl and 1.15 g/LNa₂HPO₄). 60 microliter of photoinitiator solution (300 mg of Darocur®2959, Ciba Geigy, dissolved in 700 mg ethanol) is added to themacromonomer solution and the mixture is then sterile filtered into 25ml sterile glass vial. 100 mg of sterile collagen lyophilized powder isthen added to the macromonomer solution. The dispersion is well mixedand then transferred into transparent plastic mold (cavity size 10 cm×5cm×2 mm). The dispersion is then exposed to long wavelength UV light(Blak Ray light source, model 3—100A, Flood 365 nm, intensity 30 mW/cm²)for 5 minutes. The polymerized, flexible gel is then removed from themold and used as polyalkyleneoxide-collagen hydrogel wound dressing.

10. In Situ Formation of Tissue Conformal Wound Dressing Using AqueousThermosensitive Macromonomer Solutions

10 grams of thermosensitive macromonomer (synthesized as described inexample B5) is dissolved in 20 ml cold (0-15° C.) PBS solution. To thissolution, 90 microliter of Darocur 2959 solution (300 mg in 0.7 mlethanol) is added and mixed. The cold solution is then transferred into50 ml cold syringe and filtered using 0.2 micron syringe filter intoanother vial (the solution should be cold (0-5° C.) in order to filter).The filtered cold solution is directly applied over wound surface orinjected into a wound cavity. The cold solution conforms to the woundgeometry or cavity and the body temperature causes physical gelation ofthe macromonomer. The physically gelled solution is then irradiated withlong UV light to crosslink the macromonomer solution. The light induceschemical crosslinking and forms a chemically crosslinked hydrogel whichhas good absorptive and mechanical properties. The polymerized gel isalso non-adherent to the tissue and can be easily removed from the woundsite.

It is evident from the above results and discussion that an improvedmethod of preparing protein concentrates, and particularly fibrinogenrich compositions, from blood compositions such as whole blood andplasma are provided. The subject methods are simple and easy to practiceand require less time than prior methods of preparing fibrinogen richcompositions. Furthermore, the above methods allow greater control overthe composition of the fibrinogen rich compositions than do priorpreparation methods. Importantly, the subject methods provide forprotein concentrates in which the proteins are not denatured.

All publications and patent applications cited in this specification areherein incorporated by reference as if each individual publication orpatent application were specifically and individually indicated to beincorporated by reference. The citation of any publication is for itsdisclosure prior to the filing date and should not be construed as anadmission that the present invention is not entitled to antedate suchpublication by virtue of prior invention.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, it is readily apparent to those of ordinary skill in theart in light of the teachings of this invention that certain changes andmodifications may be made thereto without departing from the spirit orscope of the appended claims.

1. A water soluble polymeric crosslinking agent comprising: an inertpolymeric component, a biodegradable component, and a branch comprisinga protein reactive functional component, wherein said crosslinking agentcomprises a plurality of branches, wherein said plurality is greaterthan two.
 2. The crosslinking agent of claim 1 wherein said crosslinkingagent is linear.
 3. The crosslinking agent of claim 1, wherein saidinert polymeric component is flanked at each end with said biodegradablecomponent which is flanked at each end with said protein reactivefunctional component.
 4. The crosslinking agent of claim 3, wherein theprotein reactive functional component is chosen from the groupconsisting of carbodiizmidazole, sulfonyl chloride, chlorocarbonates,hydroxysuccinimidyl esters, aryl halides, sulfasuccinimidyl esters, andmaleimides.
 5. A polymeric crosslinking agent for use in vivo with apatient comprising a biologically inert core attached to a polymerhaving a biodegradable component with the polymer being attached to areactive functional group capable of forming a covalent bond in waterwith at least one functional group chosen from the group consisting ofamine and thiol, wherein the crosslinking agent has at least threefunctional groups and is water soluble.
 6. The crosslinking agent ofclaim 5 wherein the biodegradable component does not contain amino acidsassembled in amino acid sequences that are enzymatically degradable whenthe crosslinker is placed in a patient.
 7. The crosslinking agent ofclaim 5 wherein the biodegradable component comprises a polymer chosenfrom the group consisting of glycoide, lactide, caprolactone, dioxanone,and trimethylene carbonate.
 8. The crosslinking agent of claim 5 whereinthe biodegradable component comprises a hydrolytically degradablechemical group chosen from the group consisting of ester, acetal,anhydride, orthoester, or disulfide.
 9. The crosslinking agent of claim5 wherein the biodegradable component comprises a polymer chosen fromthe group consisting of polyhydroxyacid, polyorthocarbonate,polyanhydride, polylactone, polyaminoacid, and polyphosphate.
 10. Thecrosslinking agent of claim 5 wherein the biodegradable component ishydrolyzable under in vivo conditions.
 11. A polymeric crosslinkingagent for use in vivo with a patient comprising a biologically inertcore attached to at least three branches that each comprise abiodegradable component hydrolyzable under in vivo conditions and the atleast three branches are each terminated with a reactive end groupcapable of forming a covalent bond in water with at least one functionalgroup chosen from the group consisting of amine and thiol wherein 1 g ofthe crosslinking agent is soluble in 100 milliliters of water.
 12. Thecrosslinking agent of claim 11 having at least four of the branches. 13.The crosslinking agent of claim 11 wherein the biodegradable polymercomprises a polymer chosen from the group consisting of glycolide,lactide, caprolactone, dioxanone, and trimethylene carbonate.
 14. Thecrosslinking agent of claim 11 wherein the biodegradable polymercomprises a polymer chosen from a group consisting of polyhydroxyacid,polyorthocarbonate, polyanhydride, polylactone, polyaminoacid, andpolyphosphate.
 15. The crosslinking agent of claim 11 having a molecularweight from 600 to 10,000 Dalton.
 16. The crosslinking agent of claim 11having a molecular weight from 600 to 100,000 Daltons.
 17. Thecrosslinking agent of claim 11 wherein the core comprises polyalkyleneoxide.
 18. The crosslinking agent of claim 17 wherein the core comprisesat least three sequential —(CH₂CH₂O)— repeats.
 19. A method of making apolymeric crosslinking agent for use in vivo with a patient comprisingactivating at least three end groups of a polymer that comprises abiodegradable component hydrolyzable under in vivo conditions andpolyalkylene oxide such that the polymer is thereby terminated withreactive functional groups that are capable of forming a covalent bondin water with at least one functional group chosen from the groupconsisting of amine and thiol.
 20. The method of claim 19 comprisingmaking the crosslinking agent to have a molecular weight from 600 to10,000 Daltons.
 21. The crosslinking agent of claim 19 having amolecular weight from 600 to 100,000 Daltons.
 22. The method of claim 19comprising choosing the biodegradable component to comprise a member ofthe group consisting of glycolide, lactide, caprolactone, dioxanone, andtrimethylene carbonate.
 23. The crosslinking agent made according to theprocess of claim
 22. 24. The method of claim 19 comprising choosing thebiodegradable component to comprise a member of the group consisting ofpolyhydroxyacid, polyorthocarbonate, polyanhydride, polylactone,polyminoacid, and polyphosphate.
 25. The crosslinking agent madeaccording to the process of claim 24.